HIV-1 cell to cell transfer across an Env-induced, actin-dependent synapse.

Jolly C, Kashefi K, Hollinshead M, Sattentau QJ - J. Exp. Med. (2004)

Bottom Line:
Receptor recognition by HIV-1 occurs via interactions between the viral surface envelope glycoprotein (Env), gp120, and CD4 and a chemokine receptor, CCR5 or CXCR4.Recruitment of these membrane molecules into polarized clusters was dependent on Env engagement of CD4 and CXCR4 and required remodelling of the actin cytoskeleton.We propose that receptor engagement by Env directs the rapid, actin-dependent recruitment of HIV receptors and adhesion molecules to the interface, resulting in a stable adhesive junction across which HIV infects the target cell.

Affiliation: The Sir William Dunn School of Pathology, University of Oxford, UK.

ABSTRACTDirect cell-cell transfer is an efficient mechanism of viral dissemination within an infected host, and human immunodeficiency virus 1 (HIV-1) can exploit this mode of spread. Receptor recognition by HIV-1 occurs via interactions between the viral surface envelope glycoprotein (Env), gp120, and CD4 and a chemokine receptor, CCR5 or CXCR4. Here, we demonstrate that the binding of CXCR4-using HIV-1-infected effector T cells to primary CD4(+)/CXCR4(+) target T cells results in rapid recruitment to the interface of CD4, CXCR4, talin, and lymphocyte function-associated antigen 1 on the target cell, and of Env and Gag on the effector cell. Recruitment of these membrane molecules into polarized clusters was dependent on Env engagement of CD4 and CXCR4 and required remodelling of the actin cytoskeleton. Transfer of Gag from effector to target cell was observed by 1 h after conjugate formation, was independent of cell-cell fusion, and was probably mediated by directed virion fusion with the target cell. We propose that receptor engagement by Env directs the rapid, actin-dependent recruitment of HIV receptors and adhesion molecules to the interface, resulting in a stable adhesive junction across which HIV infects the target cell.

fig1: Polarization of HIV-1 receptors and Env to the interface. Conjugates formed for 30 min between JurkatLAI (effector) and primary CD4+ T (target) cells were labeled by indirect immunofluorescence, fixed, and analyzed by confocal microscopy. Images are single two-dimensional x-y sections through the central region of the cells with the corresponding Nomarski view of the cell surface. (A) Cells were stained during conjugate formation for CD4 (red) and gp41 (green) with mAbs L120 and 50-69, respectively. Conjugates with receptor-Env polarization and colocalization (yellow) are indicated with arrows. Bar, 10 μm. (B) Higher magnification of a target–effector cell conjugate showing polarized CD4 (red) and Env (green) colocalized (yellow) at the interface. Bar, 1 μm. (C) CXCR4 (red) on the target and effector cells copolarizes with Env (green) on the effector cell. (D) Conjugates between target cells and uninfected Jurkats do not show polarization of CD4 (top) or CXCR4 (bottom).

Mentions:
Low-power analysis of conjugates incubated for 10–60 min at 37°C in the presence of L120 and 50-69 revealed coclustering of CD4 and Env at the interface of effector–target cell conjugates. Fig. 1 A shows a typical low-power field in which six effector–target cell conjugates can be observed, of which three reveal polarized Env and CD4. One conjugate (Fig. 1 A, left) shows strong polarization of CD4 to the interface, whereas the other two have residual CD4 around the cell periphery. Env staining of unconjugated effector cells was often clustered into one or more patches on the cell surface (Fig. 1 A). This was not due to cross-linking of Env by the mAb during live cell 37°C staining because similar patching was observed when cells were stained on ice after conjugate formation or after fixation (unpublished data). Similarly, unconjugated target cells do not show any CD4 clustering, demonstrating that mAb alone is unable to cap CD4 (Fig. 1 B). Analysis of single conjugates at higher powers confirmed that CD4 (Fig. 1 B) frequently cocapped with Env at the interface. The polarization of CD4 to the interface varied from incomplete, where clustering at the interface was observed but residual receptor was distributed around the plasma membrane, to complete, when essentially all receptor was located at the interface (Figs. 1 B, 4 A, and 5, A and B).

fig1: Polarization of HIV-1 receptors and Env to the interface. Conjugates formed for 30 min between JurkatLAI (effector) and primary CD4+ T (target) cells were labeled by indirect immunofluorescence, fixed, and analyzed by confocal microscopy. Images are single two-dimensional x-y sections through the central region of the cells with the corresponding Nomarski view of the cell surface. (A) Cells were stained during conjugate formation for CD4 (red) and gp41 (green) with mAbs L120 and 50-69, respectively. Conjugates with receptor-Env polarization and colocalization (yellow) are indicated with arrows. Bar, 10 μm. (B) Higher magnification of a target–effector cell conjugate showing polarized CD4 (red) and Env (green) colocalized (yellow) at the interface. Bar, 1 μm. (C) CXCR4 (red) on the target and effector cells copolarizes with Env (green) on the effector cell. (D) Conjugates between target cells and uninfected Jurkats do not show polarization of CD4 (top) or CXCR4 (bottom).

Mentions:
Low-power analysis of conjugates incubated for 10–60 min at 37°C in the presence of L120 and 50-69 revealed coclustering of CD4 and Env at the interface of effector–target cell conjugates. Fig. 1 A shows a typical low-power field in which six effector–target cell conjugates can be observed, of which three reveal polarized Env and CD4. One conjugate (Fig. 1 A, left) shows strong polarization of CD4 to the interface, whereas the other two have residual CD4 around the cell periphery. Env staining of unconjugated effector cells was often clustered into one or more patches on the cell surface (Fig. 1 A). This was not due to cross-linking of Env by the mAb during live cell 37°C staining because similar patching was observed when cells were stained on ice after conjugate formation or after fixation (unpublished data). Similarly, unconjugated target cells do not show any CD4 clustering, demonstrating that mAb alone is unable to cap CD4 (Fig. 1 B). Analysis of single conjugates at higher powers confirmed that CD4 (Fig. 1 B) frequently cocapped with Env at the interface. The polarization of CD4 to the interface varied from incomplete, where clustering at the interface was observed but residual receptor was distributed around the plasma membrane, to complete, when essentially all receptor was located at the interface (Figs. 1 B, 4 A, and 5, A and B).

Bottom Line:
Receptor recognition by HIV-1 occurs via interactions between the viral surface envelope glycoprotein (Env), gp120, and CD4 and a chemokine receptor, CCR5 or CXCR4.Recruitment of these membrane molecules into polarized clusters was dependent on Env engagement of CD4 and CXCR4 and required remodelling of the actin cytoskeleton.We propose that receptor engagement by Env directs the rapid, actin-dependent recruitment of HIV receptors and adhesion molecules to the interface, resulting in a stable adhesive junction across which HIV infects the target cell.

Affiliation:
The Sir William Dunn School of Pathology, University of Oxford, UK.

ABSTRACTDirect cell-cell transfer is an efficient mechanism of viral dissemination within an infected host, and human immunodeficiency virus 1 (HIV-1) can exploit this mode of spread. Receptor recognition by HIV-1 occurs via interactions between the viral surface envelope glycoprotein (Env), gp120, and CD4 and a chemokine receptor, CCR5 or CXCR4. Here, we demonstrate that the binding of CXCR4-using HIV-1-infected effector T cells to primary CD4(+)/CXCR4(+) target T cells results in rapid recruitment to the interface of CD4, CXCR4, talin, and lymphocyte function-associated antigen 1 on the target cell, and of Env and Gag on the effector cell. Recruitment of these membrane molecules into polarized clusters was dependent on Env engagement of CD4 and CXCR4 and required remodelling of the actin cytoskeleton. Transfer of Gag from effector to target cell was observed by 1 h after conjugate formation, was independent of cell-cell fusion, and was probably mediated by directed virion fusion with the target cell. We propose that receptor engagement by Env directs the rapid, actin-dependent recruitment of HIV receptors and adhesion molecules to the interface, resulting in a stable adhesive junction across which HIV infects the target cell.